Flavor is one of the most important attributes that decides the acceptability of various food items that we consume. The sensation of flavor perceived is generally because of the mixture of many chemicals in the food. Still, there are some compounds, which dominate the flavor of a particular food item and thus are themselves capable of eliciting a similar response in humans to that induced by food material. Furanones, which are found in many food products, represent one such dominating class of flavor compounds. Furanones are also important as naturally occurring flavor compounds. They are responsible for the caramel-like flavor of many fruits including strawberry, pineapple, raspberry, grapes, tomato, kiwi and mango. In addition to having a sweet and pleasant odour, furanones, especially furaneol and mesifuran, are characterized by a low odour detection threshold.
Earlier studies demonstrated that ripe mango fruits also contain high amounts of furaneol (4-hydroxy-2,5-dimethyl-3(2H)-furanone) and its methyl ether, mesifuran (2,5-dimethyl-4-methoxy-3(2H)-furanone). The fruits of cultivar Alphonso contained higher amounts of these compounds than any other cultivar and ripening of Alphonso fruits was characterized by de novo appearance and increase in the levels of these two furanones. Although furanones are not quantitatively the most dominant compounds of Alphonso fruits, the low odour detection threshold of furanones makes their contribution to Alphonso mango flavor, in terms of odour units, about 20-fold greater than that of any other volatile compound.
In spite of such crucial involvement of furanones in determining the flavor of mango and the other fruits, the biosynthesis of furaneol and mesifuran has until now been studied only in strawberry and tomato. Earlier studies on strawberry showed that out of several radiolabeled substrates fed to the ripening strawberry fruits, fructose-1,6-diphosphate had the highest rate of incorporation into furaneol. This, along with the other studies confirmed fructose-1,6-diphosphate as a natural precursor of furanones in the plants.
Further studies carried out to understand the biosynthesis of furaneol in plants indicate that fructose-1,6-diphosphate is first converted by an unknown enzyme into an unstable intermediate 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF). The furaneol forming enzyme, enone oxidoreductase which is highly similar to the NAD(P)H:quinineoxidoreductase, then reduces the exocyclic α, β unsaturated bond of HMMF, resulting in the formation of furaneol. Enone oxidoreductases from both, strawberry and tomato are capable of converting various derivatives of HMMF, substituted at the methylene group, into their respective saturated products. The presence of HMMF has also been detected in the fruits such as pineapple and raspberry suggesting that the biosynthetic pathway of furanones might be similar in different plants.
Furaneol further contributes to the fruit flavor by being converted into its methyl ether, mesifuran. The enzyme responsible for the formation of mesifuran is known only from strawberry and it was shown to be an S-adenosyl methionine dependent methyl transferase that methylates the hydroxyl group of furaneol.
An article titled “Functional characterization of enone oxidoreductases from strawberry and tomato fruit” by Klein D., published in J. Agric Food Chem. 2007 Aug. 8; 55(16): 6705-11 reports that HMMF, the substrate of FaEO that is formed during strawberry fruit ripening, was also detected in tomato and pineapple fruit by HPLC-ESI-MSn.
An article titled “Characterization of NAD(P)H-dependent Enone oxidoreductase of strawberry and tomato fruit” by Klein D., characterizes Fragaria ananassa enone oxidoreductase (FaEO) as an enzyme able to carry out two different reaction mechanisms depending on the available substrate. The enzyme from strawberry and a similar enzyme from tomato were heterologously expressed in E. coli, purified and biochemically characterized. The heterologously expressed enzyme catalyzed the formation of HDMF from D-fructose-1,6-biphosphate and NADH.
An article titled “Alternative oxidase from mango (Mangifera indica, L.) is differentially regulated during fruit ripening” by Cruz-Hernández A and Gómez-Lim M A published in Planta. 1995; 197(4):569-76, discloses analysis of alternative oxidase at the molecular level during the ripening of mango. Synthetic oligonucleotides, corresponding to conserved regions of the Sauromatum guttatum and Arabidopsis thaliana nucleotide sequences, were used as primers for polymerase chain reaction to amplify genomic DNA extracted from mango leaves. The 623-bp fragment was found to encode an open reading frame of 207 amino acids. Using this fragment one full-length cDNA clone, designated pAOMI.1, was obtained. The predicted amino-acid sequence exhibited 62, 64 and 68% identity to the S. guttatum, soybean, and A. thaliana enzymes respectively, indicating that this cDNA encodes a mango homologue of the alternative oxidase.
An article titled, “Ethylene biosynthesis and respiration during ripening in mango cultivars” by Reddy et. al. published in Indian Journal of Plant Physiology, 2001, Volume 6(4), 361-364, discloses the determination of enzymatic activities of ACC-oxidase and ACC-synthase at different stages of ripening in two varieties of mango fruits (Mangifera indica L.), viz. Amrapali and Dashehari. Among the two cultivars Dashehari showed higher level of ACC-synthase and ACC accumulation, and low level of ACC-oxidase, and ethylene production compared to Amrapali during the ripening process.
An article titled, “Expression profiling of various genes during the fruit development and ripening of mango’ by Pandit et. al. published in Plant Physiology and Biochemistry, 48 (2010) explores several flavor related genes along with a few associated to the physiology of developing and ripening in ‘Alphonso’ mango. The temporal and spatial regulation of the genes during development and ripening of ‘Alphonso’ mango has been analyzed.
As seen from the above disclosures, nucleotide sequence encoding enone oxidoreductases (EO) which play an important role in the biosynthesis of furaneol in mango is not known hitherto and there is a long standing need in the prior art for such sequences. Hence the Inventors have attempted in this research to provide artificial sequences which may be used to impart color, flavor and smell as in natural Alphonso mangoes.